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Figures

Characterisation of the cadherin-mediated junctional complex in primary
cultured cortical astrocytes. (A) Western blot analysis of the total
expression of the components of the cadherin-based junctional system in brain,
astrocytes and MDCK cells. 20 μg aliquots of homogenates were loaded onto
11% SDS-polyacrylamide gel and immunoblotted with the indicated antibodies.
The MDCK cell homogenate was probed with the E-cadherin antibody, and the
brain and astrocyte homogenate with the pan-cadherin antibody. (B) Affinity
chromatography. The rat cortical astrocyte lysate was incubated with
immobilised GST-LIN-7A fusion protein. The bound material (Bound) was resolved
on 10% SDS-PAGE and immunostained for β-catenin. 10% of the total
astrocyte lysate (Lys) used in the experiment was probed with the same
antibody. (C) Co-immunoprecipitation of β-catenin with the LIN-7
antibody. The rat cortical astrocytes were extracted in lysis buffer and
immunoprecipitated with the LIN-7 antibody (IP: LIN-7) or the pre-immune serum
(IP: Pre). The immunoprecipitates were loaded onto 11% SDS-PAGE and
immunoblotted with the β-catenin and LIN-7 antibodies. Molecular weight
standards expressed in kDa are indicated on the left (A-C). (D) Confocal
analysis of double immunofluorescence staining for LIN-7 and β-catenin in
cultured primary astrocytes. The astrocytes were plated onto
poly-L-lysine-coated glass coverslips, cultured until they reached confluence
(recently confluent) or had been confluent for a longer period (long
confluent) before fixation in 4% paraformaldehyde. Bar, 10 μm.

Biochemical characterisation of the cadherin-mediated junctional complex in
the T98G and U373MG glioblastoma cell lines. (A) In vitro cell migration assay
of T98G and U373MG cells. Serum-free medium containing 0.1% BSA (basal) or
conditioned media from the same cells (stimulated) were used as
chemoattractants. The data are expressed as the mean number of migrating cells
per field±s.d. for triplicate samples from a representative experiment.
(B) Western blot analysis of total cadherin, β-catenin and LIN-7
expression in glioblastoma cell lines grown to confluence. 50 μg aliquots
of total glioblastoma cell homogenates were loaded onto 11% SDS-PAGE and
immunoblotted with the indicated antibodies. (C) Surface biotinylation assay.
After being grown to confluence, the glioblastoma cell lines were surface
biotinylated with NHS-ss-biotin. After cell lysis, the surface-biotinylated
proteins were recovered using streptavidin beads, loaded onto 10% SDS-PAGE and
immunoblotted with the indicated antibodies. The results were
densitometrically analysed using the NIH Image 1.61 programme. The
corresponding β-catenin/N-cadherin ratio (expressed as relative units) is
shown on the right. (D) Affinity chromatography. Confluent cell lysates were
incubated with immobilised GST-LIN-7A fusion protein: the bound material
(Bound) was resolved by 10% SDS-PAGE and immunostained for β-catenin. (E)
Co-immunoprecipitation of β-catenin with the LIN-7 antibody. Lysates of
confluent cells were immunoprecipitated with the LIN-7 antibody (IP), and the
immunocomplexes were resolved by 11% SDS-PAGE and immunoprobed forβ
-catenin. 10% of the total T98G or U373MG lysate (Lys) used in the
experiments was probed with the indicated antibody (D-E). Molecular weight
standards expressed in kDa are indicated on the left (B-E).

Biochemical analysis of the degree of maturation of cell-cell contacts in
T98G and U373MG cell lines. (A) Western blot analysis of the amount of LIN-7
and β-catenin recovered in the TX-100-soluble (S) and TX-100-insoluble
(I) fractions. T98G or U373MG glioblastoma cells grown to confluence were
extracted in TX-100. Equivalent volumes of the soluble or insoluble fractions
were separated by 10% SDS-PAGE and immunostained using the indicated
antibodies. (B) Total P-tyr content in T98G and U373MG cells.
5×105 cells were plated, cultured for 72 hours and extracted
in lysis buffer containing 0.02% SDS and 100 μM pervanadate. Five percent
of the cell lysate used in the immunoprecipitation experiments was probed for
P-tyr. (C,D) Tyrosine phosphorylation of α- and β-catenin. The cell
lysates were immunoprecipitated with anti-α-(C) or anti-β-catenin
(D) antibodies and probed for P-tyr. To confirm similar protein loading, 10%
of the immunoprecipitates were probed for α- (C) or β-catenin (D).
Molecular weight standards expressed in kDa are indicated on the left.

Analysis of cell-cell adhesion in invasive T98G cells. (A) Matrigel
invasion assay using as chemoattractants either serum-free medium containing
0.1% BSA (basal) or conditioned media from the same cells cultured in
Matrigel-coated Petri dish (stimulated). The data are the mean number of cells
per field±s.d. of triplicate samples from a representative experiment.
(B) Western blot analysis of cadherin, β-catenin and LIN-7 total
expression in T98G cells cultured on poly-L-lysine (lane 1) or Matrigel (lane
2). The same amounts of total cell homogenates were loaded onto 11% SDS-PAGE
and immunoblotted with the indicated antibodies. Molecular weight standards
expressed in kDa are indicated on the left. (C) Brightfield and (D) confocal
laser microscopy of T98G cells cultured in Matrigel. The cells
(2×105 cells/35 mm diameter dish) were seeded onto
Matrigel-coated glass coverslips and cultured for three days. After fixation
in 4% paraformaldehyde, the cells were double-stained for β-catenin and
the indicated antibodies. The arrows indicate β-catenin-enriched
cell-cell contacts with disorganised filaments of F-actin, virtually no LIN-7
and the accumulation of phosphorylated proteins. Bar, 10 μm.

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